Biospecific Affinity Chromatography has proven to be of great value in the isolation of important biological macromolecules. This project seeks to examine details of the molecular interactions involved in binding of protein species to immobilized ligands. By adding soluble ligand components to the eluting buffer it is also possible to examine binding of these ligands to the protein by the effect on protein elution volume. These experiments will provide some unique information in addition to the measurement of protein-soluble ligand dissociation constants. First, the interaction of a protein with an immobilized ligand may involve different types of binding including hydrophobic effects of binding to parts of the matrix structure. Our results will allow an examination of these effects which could lead to redesign of affinity matrices to provide greater specificity. Secondly, the physical arrangement provides the opportunity to investigate secondary interactions on proteins. Finally, we plan to extend this approach to other biological interactions such as protein-protein binding and systems that might exhibit cooperativity or drug-protein or drug-receptor binding.